Prolactin (PRL) is a peptide hormone from the anterior pituitary gland that participates in the regulation of a variety of targets. Its modus operandi is the subject of this research. The prevailing concept in this area which had previously guided the formulation of questions and thus experimental design, was that peptide hormones, as "first messengers", cannot enter their target cells and, therefore, that the operation of "second messengers" generated from an interaction of the hormone exclusively with the target plasmalemma must be invoked. In 1976, we demonstrated unequivocally that not only can PRL enter its major target, the milk secretory cell, (MSC), it does so physiologically. Furthermore, during the course of its intracellular sojourn, it becomes intimately associated with MSC nuclei. Thus, an alternative to the "second messenger" hypothesis of peptide hormone action was born. The major objective of our present work is to continue to examine this alternative, namely, that PRL regulation requires its direct contact with subcellular constituents. Our efforts during this particular period of support are directed along two major lines. One involves an analysis of the inter- and intracellular distribution patterns of enzymatic and secretory protein markers of hormone action in relation to distribution patterns of not only homologous, but endogenous, i.e., the animal's own, PRL. This is being done in rat and cow mammary tissue with immunohistochemical and biochemical cell separation and fractionation techniques. The other deals with the possibility of the existence of "clipzymes" specific of target cells that would cleave the large, multiple-action PRL molecule into single-action fragments. This hypothesis could explain both a requirement for target cell incorporation and the pluritrophic nature of this hormone. To study this, we have been investigating possible immunoreactive homology betwwen PRL and a small peptide (MW less than 2000) with PRL-like activity in rat ovarian follicles, using immunohistochemistry. With a mammary gland explant system and cell fractions, we plan to test this peptide further, both for PRL-like bio- and immuno-activity. The significance of this research is, ipso facto, speculative at this moment. It is in retrospect that it must be evaluated.